At the Cutting-Edge of Regenerative Medicine: Bioengineering Human-Sized Bone

Fisher et al published the first attempt at engineering

a full-scale adult human femur head from hMSCs.  This

is the largest reported tissue to have been engineered

and took over 700 million hMSCs to fabricate.

John Fisher’s laboratory at the University of Maryland, College Park recently published what can be considered a significant advance for Tissue Engineering and Regenerative Medicine. Graduate student Bao Nguyen and her colleagues have engineered a bone construct that is 20 times larger than any reported previously, and the size of an adult human femur.

Why is this important? Critical size bone defects are a significant health problem (resulting in over $1 billion in annual healthcare costs incurred in the U.S.) and are currently treated with grafts, decellularized bone, or synthetic bone grafts, with sometimes unsucessful results. As such, modern medicine has been looking to tissue engineered bone grafts as future treatments for such defects. Human bone marrow-derived Mesenchymal Stem Cells (hBM-MSCs) are a promising cell source for such applications because they efficiently differentiate down the osteogenic path and also secrete paracrine factors that may aid survival and vascularization of engineered bone. Prior to this publication, engineered constructs have been relatively small due to cell and culture limits.  One major challenge has been growing hBM-MSCs, while maintaining their function, to sufficient numbers needed for an adult human-sized construct; a challenge adressed by RoosterBio.  In addition, nutrient and O₂ transfer are often insufficient to maintain cell viability and function throughout larger constructs, especially those of adult human dimension.

To address this cell culture limitation, the Fisher laboratory developed a Tubular Perfusion System (TPS) bioreactor where cells and scaffolds are cultured in a cylindrical chamber and subject to circular media flow. This system has high nutrient and O₂ transfer and efficient waste removal and has been previously used to produce smaller engineered bone and cartilage constructs (See here and here).

In the study detailed here, the authors had access to and combined, for the first time, advanced technologies required for biofabrication: 3D printing, the TPS bioreactor, and scalable production of hBM-MSCs. The goal of the study was to scale-up bone constructs to adult human size. A full size mold of the superior portion of a human femur (the largest bone in the human body) was 3D printed using information from an opensource database. The mold measured 23 cm long and 10 cm at its widest point with a volume of  200 cm³. The mold was filled with hBM-MSCs in alginate beads (3 mm beads, 100,000 cells per bead). The entire construct utlillized 7200 aliginate beads containing a total of 7.2 x 10⁸ cells (yes, that is 720 million cells!). The high volume hBM-MSC cell and media systems used were from RoosterBio, and technical support for the efficient production of large volumes of hBM-MSCs was provided by our company.

After 8 days of culture in the TPS, the construct was examined for cell viability and bone differentiation. High cell viability was seen in all parts of the construct, both on the outside and the inside (interior). In addition, hBM-MSCs committed to the osteogenic lineage throughout the construct, demonstrating efficiency of the TPS culture system. Both early (Alkaline Phosphotase, ALP) and late (Bone Morphogenic Protein-2, BMP-2) markers of osteogenesis were upregulated relative to day 0. Interestingly, ALP and BMP-2 expression was 25- to 30-fold higher in the construct shaft relative to other portions of the construct. The authors speculate that this is due to shear stress exerted on the parts of the construct closest to the inlet, which activates hBM-MSC signaling pathways, causing release of paracrine factors that stimulate osteogenesis of the “downstream” shaft portion. Taken together, these results demonstrate that the confluence of cutting-edge technologies such as 3D printing, TPS bioreactors, and best-in-class hBM-MSC manufacturing processes enable the engineering of adult human-sized tissue constructs.

While “…this first foray into full-scale bone engineering provides the foundation for future clinical applications of bioengineered bone grafts…” the authors point out some limitations to this study. The 8 day culture period was relatively short, given the weeks usually needed for high efficiency bone differentiation. Thus, extended time points and the fabrication of additional large constructs are needed to fully explore the capabilities of the TPS system. Further, alginate is a soft material, and its mechanical properties do not render it the best suited for bone differentiation.  In addition, hBM-MSCs within aliginate beads lack cell:cell contact, which may also limit their osteogenic differentiation.  To address these limitations of the current system, the Fisher group is developing a 3D printed shell made of an implantable rigid material better suited for the engineering of bone constructs.  Finally, the construct lacks a vascular network, which can be overcome by including endothelial cells (EC) in addition to hBM-MSCs or by incorporating micro-channels in the engineered constructs through a variety of methods (e.g. biomaterial fabrication and 3D printing). Despite the aforementioned limitations, the work presented is a significant advance towards clinical-sized tissue-engineered bone constructs for use in patients.

In an attempt to elicit discussion, I will mention other methods that harbor potential for use in such large-scale tissue engineering applications. For one, hBM-MSC aggregates could be used in place of cells in alginate beads. These 3D-MSC not only maintain cell:cell contact but also undergo osteogenic differentiation more efficiently than cells grown on tissue culture plastic, are resistant to hypoxia, and secrete angiogenic cytokines. Secondly, factors that stimulate bone differentiation of hBM-MSCs, and/or alter mechanical properties of the construct, could be incorporated into the polymer scaffolding, or could be introduced into 3D-MSC aggregates. Finally, once bio-inks are developed further, the bone construct could be patterned by 3D printing of cells (hBM-MSC, EC, 3D-MSC) and materials.  Now that human-sized constructs are possible in terms of cell numbers and O₂ and nutrient diffusion, the possibilities are virtually endless.

Finally, we sincerely thank Bao Nguyen and John Fisher for being early adopters of RoosterBio hBM-MSCs and joining us in accelerating Regenerative Medicine!

References:

Nguyen BB, Ko H, Moriarty RA, Etheridge JM, Fisher JP. Dynamic Bioreactor Culture of High Volume Engineered Bone Tissue. Tissue Engineering Part A. Volume 22, Numbers 3 and 4, 2016, ahead of print. doi:10.1089/ten.tea.2015.0395.  http://online.liebertpub.com/doi/abs/10.1089/ten.tea.2015.0395 

I’m sorry that this is paywalled!

Yeatts, A.B., and Fisher, J.P. Tubular perfusion system for the long-term dynamic culture of human mesenchymal stem cells. Tissue Eng Part C 17, 337, 2011.

http://online.liebertpub.com/doi/abs/10.1089/ten.tec.2011.0263

Yeatts, A.B., Choquette, D.T., and Fisher, J.P. Bioreactors to influence stem cell fate: augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems. Biochim Biophys Acta 1830, 2470, 2013.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461086/

Ma, X et al. Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting  PNAS, Early Edition doi: 10.1073/pnas.1524510113 http://www.pnas.org/content/early/2016/02/04/1524510113

An Open Letter to the Builders of the Cell-based BioEconomy

Dear Stem Cell Pioneer:

February marks the one year anniversary of RoosterBio shipping our first stem cell products to our valued customers, and I personally am very excited for the coming year ahead. We look forward to delivering even more high quality stem cells to people like you that are doing amazing things.

Looking forward into 2015 and beyond, I want to make sure we are staying true to our mission: to greatly increase the availability and accessibility of stem cell technology to researchers and product developers across the globe – and that we are committed to our vision of accelerating the pace of product development in the cellular therapy, bioprinting and tissue engineering markets.  I am hoping to focus our efforts to making sure that we are moving your discoveries and developments forward faster than anticipated.  This will not just be a win for our customers and RoosterBio, but for the entire Regenerative Medicine field.

In 2014 after launching our hMSCs in the unprecedented product format of 10 million cell vials to glowing reviews, we quickly implemented our Starter Kits and Working Cell Bank formats based on your feedback.  These new formats allow for accelerated testing, performance verification, and standardized small scale experimentation with reproducible outcomes.  We also initiated multiple collaborations with leaders in the tissue engineering and biofabrication fields – which has led to multiple conference posters, presentations, and soon to be submitted publications.

We also find ourselves at the precipice of a boom in biofabrication technologies, and we consider this the beginning of the Golden Age of Tissue Engineering.  I anticipate great progress will be made at an increasingly rapid pace.  Now that many of the tools required for bioprinting are becoming “democratized” (simpler, less expensive, more accessible) such as 3D BioPrinters, biomaterials, and primary cells – laboratories can get up and running in a matter of weeks with limited initial resources, something that would have taken months to years and extensive capital in the past.  We are at a special time, and the entire field will be accelerating forward at a rapid pace, making biofabrication truly an exponential medical technology.

2015 will truly be an exciting year for RoosterBio.  We will be participating in a Stem Cell Manufacturing Training Program, helping to organize several conferences on Cell Therapy BioProcessing and BioPrinting, exhibiting at multiple conferences, as well are contributing to initiatives such as the Georgia Tech Cell Manufacturing Consortium and the NIST Workshop on Strategies to Achieve Measurement Assurance for Cell Therapy Products.  The primary motivation behind these initiatives are to make sure that we are driving forward our vision and delivering on our mission.

None of this would be possible without the hard work and dedication of the entire RoosterBio team, as well as the support that we are getting from you, our valued customers.  Please continue to join us on our journey as we accelerate the development of the Cell-based BioEconomy.

All the best from Frederick, Maryland.


Jon A Rowley
Chief Executive & Technology Officer
RoosterBio Inc.

Welcome to the Golden Age of BioPrinting, Tissue Engineering and BioFabrication

A"Golden Age" is defined as a period of time in a field where "great tasks are accomplished."  The ancient Greek philosopher Hesiod initially coined this phrase, and I think if he were alive today, he would agree with us that we are in a special time of technology convergence where innovations and advancements are progressing at an accelerating rate.  The fields of Tissue Engineering and Regenerative Medicine are benefiting from these rapid technology advancements.



We are now at the beginnings of the Golden Age of BioFabrication.  The last 20 years has seen steady progress in the Tissue Engineering field, but the cost and time it has taken to develop products based on these technologies has been prohibitive.  Thus, only the best funded labs have been able to perform this very expensive R&D.  Within the last year, products such as high volume stem cells (via RoosterBio) and low cost bioprinters (from our collaborators BioBots) have been coming to market and dramatically reduce the cost, the time, and the complexity to fabricate three dimensional biological structures that are the precursors to tomorrow's tissue engineered products.  By removing the technology and cost barriers and democratizing biofabrication technology, more labs can now afford to do the applied R&D, allowing more work to be accomplished faster, completely changing the equation of how labs function.  This is accelerating the development of this entire field.

 Walter Isaacson makes the point over and over in his new book The Innovators that collaboration between people and groups with complementary skill sets is essential to innovation and technology progress.  We, at RoosterBio, have always said that communication platforms (such as social networks, conferences, biohacker spacers, blogs, and journals) are also critical for those in a field to share knowledge and experiences - further progressing the thought convergence.  This February 9th and 10th in Boston is a focused conference on Tissue Engineering and BioPrinting that SelectBio is hosting.  The top researchers, thought leaders, and product developers in the field will be presenting cutting edge research, technology development, and commercialization strategies.  We hope to see you there.

Regenerative Medicine Standards Development – an FDA Workshop and an Initiative in Need of Structure

As we stated in an earlier blog post, standardization of materials, equipment and processes will be critical to drive reproducibility and robustness of living cell technologies to the point where they can be widely used and considered “Democratized”.  Some day in the future, an advanced degree and several years of training will not be a pre-requisite for a person to use living cells for some type of particular task, be it for personal or professional applications.  Since standards are so important to RoosterBio’s long-term mission of Democratizing Cellular Technologies, I devoted my March 31^st to attending an FDA-hosted public workshop titled Synergizing Efforts in Standards Development for Cellular Therapies and Regenerative Medicine.  The agenda for the day can be found here.

There has been a robust ongoing effort forged between several academic and industry groups (ISCT, ARM, TERMIS) and standards-related organizations (NIST, ASTM, ISO, AABB, ICCBBA, USP, FACT) to establish standards for cell therapy and regenerative medicine; enough that the FDA thought it wise to begin to try to organize and coordinate these activities.  Thus, the purpose of this workshop was to “bring together a broad range of stakeholders to discuss current and future standards development activities involving cellular therapies and regenerative medicine products”.  While there wasn’t a lot of “action” at this meeting, it was a very good mechanism for understanding the landscape of the various activities, who some of the key players are, and how to get actively involved in the dialogue.

Democratizing Living Cellular Technology

@JennWebb recently wrote an article for the O’Reilly Radar titled Democratizing Technology and the Road to Empowerment.  She starts out the article with a nice summary of what it means to Democratize Technology.  Jenn writes “Advancements in technology are making what once was relegated only to highly educated scientists, engineers and developers accessible to — and affordable for — the mainstream.“  Now, the blog she writes for is focused on the intersection of Hardware and Software (or the “physical and digital worlds” is how they phrase it), while we at RoosterBio are imagining a World where biotechnology, specifically living cellular technologies, are simplified and cost-reduced to the point that you don’t have to be a PhD researcher in a well-funded laboratory to perform your own experiments or build novel things out of living cells. The concept of biology paralleling the advances of IT are well laid out elsewhere.

Today, it is much easier to incorporate living cells into your research than it was 20 years ago.  This is evidenced by the proliferation of Cell Biology capabilities in Engineering departments all over the world as Biomedical Engineering has turned into a formalized academic discipline.  When I was doing undergraduate research at the University of Michigan in the early 1990’s, it took months and several collaboration attempts before we could get living cells onto the biomaterial constructs we were making at the time.  Today, it is more commonplace to find the tools to marry the Worlds of Cell Biology and Engineering in the same laboratory.  Despite this, the total number of labs with such capabilities and expertise is still very small.

 We believe that the steps required to fully Democratize Cellular Technologies will be to:

Welcome to the RoosterBio Blog

Everyone at RoosterBio is extremely excited to be launching our company, shipping our first products, and interacting with tissue engineers, cell therapists, synthetic biologists, and our customers to learn about innovative research in these fields, share our knowledge, and contribute to the Cell and Tissue Engineering Revolution. The central theme behind RoosterBio, or what we call our “business hypothesis”, is that as living cellular technologies become more affordable, easier to access, and much simpler to incorporate into product development efforts– there will be a rapid acceleration in products coming to market that incorporate these technologies. We believe that we can help shape this new market by Democratizing Cell Technologies and making them abundant, affordable, and much simpler to translate into the clinic.

We have assembled a team at RoosterBio that have years of experience in stem cell R&D, cell therapeutic product and process development, and manufacturing operations, and with this blog we hope to share many of the topics that we are so enthusiastic about.  We gravitate toward technologies at the technical interfaces of fields such as 3D printing and tissue engineering; thus 3D bioprinting of tissues will be a big topic on this blog.  We will be sharing our thoughts on step-changes in technology on topics like stem cell therapies, engineered tissues and organs, biological robots, manufacturing technologies, and synthetic biology.  We will also engage in educational posts related to our expertise, and comment on broad themes that are facing the industries that we care about, such as establishing standards in the stem cell arena.  The developments in progressive fields always have profound effects on science and society, and we believe we are on the verge of the cell and tissue engineering revolution. 

This blog is targeted at the scientists, technologists, engineers, and doctors that are working passionately and feverishly to bring cell-based products to patients, as well as for general audiences that are interested in the driving forces behind cellular therapies, regenerative medicines, and tissue engineering technologies.  We hope you will come back to learn and engage in the conversation, and also be a part of the next technology revolution as Biology becomes Technology. 

We encourage everyone to leave comments, and to feel free to say what is on your mind. We look forward to the dialogue and helping to accelerate the Cell-based BioEconomy!

- The RoosterBio Team